Madin-Darby canine kidney cells accumulate several nonperturbing organic osmolytes when cultured in a hypertonic medium. Myo-inositol, betaine, and taurine are accumulated secondary to an increase in uptake, the first coupled to sodium entry, the latter two coupled to sodium and chloride entry. The transport rates increase as the result of an increase in maximum velocity for each cotransporter, with peak activity 24 h after the increase in tonicity. The cDNA for each cotransporter has been cloned. Their sequences indicate that the myo-inositol cotransporter belongs to the gene family that includes the sodium-coupled glucose transporter (SGLT1); the betaine and taurine cotransporters belong to the gene family of sodium- and chloride-coupled transporters that are responsible for neuronal uptake of many neurotransmitters. Assays of mRNA abundance and nuclear run-on assays reveal that shifts in tonicity have a major effect on transcription of the genes for the sodium-myo-inositol (SMIT) and sodium-chloride-betaine (BGT1) cotransporters. The ensuing increase in mRNA abundance for the two cotransporters and presumed increase in synthesis of the cotransporter proteins can explain the increase in transport activity in response to changes in tonicity.